Exploring the potential of Bornean polypore fungi as biological control agents against pathogenic Ganoderma boninense causing basal stem rot in oil palm

Basal stem rot due to a fungal pathogen, Ganoderma boninense, is one of the most devastating diseases in oil palm throughout the major palm oil producer countries. This study investigated the potential of polypore fungi as biological control agents against pathogenic G. boninense in oil palm. In vitro antagonistic screening of selected non-pathogenic polypore fungi was performed. Based on in planta fungi inoculation on oil palm seedlings, eight of the 21 fungi isolates tested (GL01, GL01, RDC06, RDC24, SRP11, SRP12, SRP17, and SRP18) were non-pathogenic. In vitro antagonistic assays against G. boninense revealed that the percentage inhibition of radial growth (PIRG) in dual culture assay for SRP11 (69.7%), SRP17 (67.3%), and SRP18 (72.7%) was relatively high. Percentage inhibition of diameter growth (PIDG) in volatile organic compounds (VOCs) in dual plate assay of SRP11, SRP17, and SRP18 isolates were 43.2%, 51.6%, and 52.1%, respectively. Molecular identification using the internal transcribed spacer gene sequences of SRP11, SRP17, and SRP18 isolates revealed that they were Fomes sp., Trametes elegans, and Trametes lactinea, respectively.


Results
Pathogenicity of polypore fungi. To distinguish between the pathogenic and non-pathogenic fungi, disease signs and symptoms in the tested oil palm seedlings, indicating successful infection of the polypore fungi were observed and recorded at week four post inoculation. The results are shown in Fig. 1. Among the 21 polypore fungi isolates tested, 12 of them caused infection with typical disease signs and symptoms, including the appearance of chlorotic leaves, necrotic leaves, fungal mass on the oil palm seedling bole, and necrotic roots after four weeks of incubation. The expressed disease signs and symptoms were similar in all the infected seedlings, regardless of the polypore fungi isolates tested. Uninoculated seedlings, seedlings inoculated with two non-pathogenic Ganoderma lucidium isolates and six other polypore fungi isolates remained symptomless throughout the experiment. Table 1 depicts the degree of infection by G. boninense in oil palm seedlings based on disease incidence, disease severity, percentage of symptomatic leaves, and percentage of infected primary roots. Oil palm seedlings inoculated with the polypore fungi G. lucidum (GL01 and GL02), RDC06, RDC24, SRP11, SRP12, SRP17, and SRP18 showed no disease incidence and were healthy, similar to the uninoculated negative control. These fungi isolates were deemed non-pathogenic and were chosen for subsequent in vitro antagonistic assays.
The ability of the fungi to inhibit G. boninense in the dual culture and VOCs dual plate assays were not significantly correlated (p = 0.38, r = 0.33). Also, SRP11 and SRP18 recorded the highest PIRG in the dual culture assay at 67.7% and 72.7%, respectively, followed by SRP17 (67.3%). Similarly, SRP11, SRP17, and SRP18 recorded the highest PIDG in the VOCs dual plate assay at 43.2%, 51.6%, and 52.1%, respectively. Therefore, the three isolates (SRP11, SRP17, and SRP18) were selected for further investigation as potential candidates of BCAs against G. boninense in oil palm (Fig. 2). Based on the ITS sequences, the isolates of SRP11, SRP17, and SRP18 were identified as Fomes sp., Trametes elegans, and Trametes lactinea, respectively, with 99 -100% homology with the deduced nucleotide fragment sequences searched using BLAST (Table 4).

Discussion
Typical Ganoderma infection was observed on the oil palm seedlings. The infection was initiated at the root since the roots and inoculum source were in direct contact. The infection later progressed to the stem bole, mainly through the inner thin-walled cortex. Then, foliar symptoms (chlorosis and necrosis) appeared as a result of the damaged internal stem bole or disruption of the xylem and phloem resulting in the water and nutrients required for the seedlings not being sustained, which caused progressive desiccation of the leaves from the oldest to the youngest. The infected seedlings eventually died, either with or without visible fungal masses on the seedlings above ground 23,24 . Furthermore, the in planta infection of oil palm seedlings in this study adopted from Purnamasari et al. showed similar disease progression over four weeks 25 , compared with those infected using the technique of placing rubber wood block with Ganoderma as inoculum on older oil palm seedlings, i.e. 3-months-old, and data collection carried out over six months 23 www.nature.com/scientificreports/ The different levels of aggressiveness of the polypore fungi tested could be related to the modes of nutrition acquisition strategies during fungal pathogen infection on the plants, which have traditionally been classified into biotrophic and necrotrophic types of parasitism 27 . The highly pathogenic isolates identified in this study, SRP01 and SRP14, as well as the positive control G. boninense (G4), were necrotrophs, which are facultative parasites that derive energy from a living host, kill the host's cells, and then live saprophytically on the dead remains 28 . According to Idris et al. 29 and Castillo et al. 30 , three species of pathogenic Ganoderma, namely, G. boninense, G. zonatum, and G. miniatocinctum were associated with BSR in oil palm. Other species, such as G. tornatum, G. applanatum, G. lucidum, G. pfeifferi, and G. philippi, were not pathogenic 31 . Meanwhile, the least infection in the isolates RDC07, RDC30, SRP02, and SRP08 could be related to biotrophic facultative saprophytic parasitism,     28,32 . The polypores, RDC24, SRP11, SRP17, and SRP18 in this study demonstrated strong antagonistic activity against G. boninense (G4) based on the dual culture assay, with a PIRG value of 61.2% to 72.7%. The findings in this study are similar to that reported by Naidu et al., where the in vitro PIRG value of G. boninense using the same assay was between 70 and 80% 22 . The fungi Pycnoporus sanguineus, Trametes lactinea, and Grammothele fuligo inhibited the in vitro growth of G. boninense by 84%, 82%, and 81%, respectively. Furthermore, another study by Naidu et al. also reported that in vitro antagonism of G. boninense using the same assay was between 73 and 85%, where Neonothopanus nambi exhibited high antagonism 33 . In general, the inhibition of G. boninense could be due to the predominance of BCAs against the pathogen due to competition for the available nutrients and space, parasitism, the secretion of antibiotics, or the production of secondary and volatile metabolites, and enzymes 7,19,34 . Non-volatile metabolites (e.g., alkaloids, flavonoids and fatty acids), and volatile metabolites (e.g., aromatic metabolites, terpenes, polyketides, butenolides and pyrones) are among the metabolites that have antifungal activity against fungi 35,36 . These metabolites may inhibit ergosterol synthesis (the main fungal sterol) and macromolecular synthesis, and be toxic to phytopathogens 37 . In addition, hydrolytic enzymes such as the chitinases produced by BCAs are able to degrade the fungal cell wall of phytopathogens, which further increases the permeability of the cell wall to subsequent entry of antifungal metabolites. Furthermore, several proteases from BCAs have been reported to act on the phytopathogens' enzymes and impair their functions 19 . Several studies reported that the secondary metabolites released by Trametes spp. showed antifungal properties with inhibitory effects on complex phytopathogenic fungi such as G. boninense 38,39 . Moreover, it was reported that the fruiting bodies of Trametes spp. contained phenolic and flavonoid compounds and other chemicals, such as saponin, tannin, and alkaloid, giving them antifungal properties 39,40 .
Although the potential of Trametes lactinea as a BCA to suppress G. boninense in oil palm has been previously reported 22,29 , there is still a lack of information available on the role of Trametes species as BCAs against crop diseases. In particular, Trametes versicolor was the most dominant species of this genus in controlling the tomato pathogens Clavibacter michiganensis, Ralstonia solanacearum, and Leveillula taurica 41,42 (Table 5). However, the role of T. elegans and Formes sp. as BCAs in controlling G. boninense or other phytopathogens remains unknown.
The Bornean non-pathogenic polypore fungi discovered in this study need to be further characterized, such as by evaluating their metabolite production using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), for better understanding of their roles in exerting antagonism towards G. boninense. Furthermore, in planta suppression of G. boninense infection utilizing the possible antagonists discovered, either at seedling, immature, or mature stages of oil palm, will require longer-term observation and data collection to corroborate the findings.

Materials and methods
Pathogenicity of polypore fungi. A total of 150 germinated oil palm seeds (a cross between Deli dura and AVROS pisifera) were acquired from Sawit Kinabalu Balung, Tawau, Sabah, Malaysia (4.2667° N, 117.9631° E), a commercially certified oil palm seed producer. The germinated oil palm seeds were sown in three planting pot trays (50 pots per tray) containing topsoil. The seedlings were grown in a rain-shelter facility at the Faculty of Sustainable Agriculture of Universiti Malaysia Sabah (5.9300° N, 118.0112° E) for three months, with standard nursery management practices 43 . These seedlings were used for the pathogenicity experiment. The pathogenicity of 21 polypore fungi initially isolated by Darwana et al. 44 was used in this study, to evaluate whether they were pathogenic or non-pathogenic towards oil palm. The fungi were isolated from the basidiocarp using potato dextrose agar (PDA) containing 50 mg L −1 streptomycin to prevent the growth of bacteria 45 . The pure cultures of the fungi were confirmed as dikaryotic basidiomycetes based on the presence of hyphal septa and clamp connections under microscopic observation. The fungi used in this study were obtained from its stock culture on PDA slant stored in 4 °C temperature.
The positive control comprised of G. boninense (G4) isolated from a BSR-infected oil palm, while two G. lucidum isolates (GL01 and GL02) served as the non-pathogenic internal control. The taxonomic identity of G4, GL01, and GL02 had been previously verified based on their genomic DNA fragment sequences (Supplementary Appendix S1). Other polypore fungi samples were collected from the Rainforest Discovery Centre (RDC; 5.8746° N, 117.9436° E) and Sandakan Rainforest Park (SRP; 5.8511° N, 118.0642° E) in Sandakan, Sabah, Malaysia. Samples were denoted by their local origin, i.e., RDC or SPC, followed by sample number.
Fungal inoculums preparation and oil palm seedling inoculation were prepared according to the method of Purnamasari et al., with minor modifications 25 . Specifically, a seven-day-old active mycelial plug (10 mm in diameter) was cultured in an Erlenmeyer flask containing 150 mL of potato dextrose broth (PDB) and incubated at 28 °C for five days on a shaking incubator at 150 rpm. The mycelia were filtered and transferred into a beaker containing 200 mL of sterile distilled water and 0.002% Tween 20. The mycelia was then blended using a 70% ethanol-sanitised handheld blender (Panasonic MX-SS40) for three minutes. The concentration of mycelia was adjusted to 10 5 fragments mL −1 using a hemocytometer.
Oil palm seedlings were carefully removed from the pot tray, and excessive soil was loosened from the roots before soaking in the fungi inoculum for 30 min. Uninoculated seedlings soaked in sterile distilled water served as the control. All seedlings were transplanted into polybags (15 cm × 30 cm) containing 600 g of sterilised topsoil with five replications per treatment, yielding 110 experimental units. They were arranged based on a completely randomised design (CRD) in an environmentally regulated laboratory at 25-28 °C temperature, 70-80% relative humidity, and sufficient sunlight. The seedlings were watered twice daily with sterile distilled water to maintain soil moisture.
The presence of the pathogen (disease sign) and disease symptoms were recorded four weeks post inoculation. These disease signs and symptoms included disease incidence, disease severity, the total number of leaves, the number of symptomatic leaves, the total number of primary roots, and the number of infected primary roots 23 . The visually observable symptoms of infected seedlings was based on numerical values of disease class ranging from 0 to 4 (Table 6) 46 . The numerical values were transformed to a percentage of disease severity using Eq. (1). The procedures involving plant samples were performed in accordance with the relevant guidelines and regulations, including autoclaving the samples to eliminate all possible biological contamination before disposal of the samples.
(1) Disease severity (%) = Disease class of seedling The highest disease class × 100  www.nature.com/scientificreports/ In vitro antagonistic dual culture assays. The antagonistic effect of selected non-pathogenic polypore fungi against G. boninense was evaluated using the dual culture assay of Rahman et al. 47 . Mycelia plugs (8 mm in diameter) of the potential antagonist and pathogen (G4) were inoculated 20 mm away from the edge of a Petri dish containing PDA (90 mm diameter) on opposite sides. A PDA plate without an antagonist served as the control. The experiment was performed with three replications, arranged in a CRD, incubated for seven days, at 28 °C, and in darkness. The percentage inhibition of radial growth (PIRG) was calculated using Eq. (2) 48 .
where, R1 is the colony radius of G. boninense (G4) isolates in the control plate and R2 is the colony radius of G. boninense (G4) isolate in the dual culture assay. The inhibition zone formed between the colonies was also observed. The antagonistic effect of each isolate against the growth of the G. boninense (G4) was classified as very weak (1-20%), weak (21-40%), moderate (41-60%), strong (61-80%), and very strong (81-100%) based on their PIRG values.
In vitro antagonistic volatile organic compounds (VOCs) dual plate assays. An assay of VOCs was performed to evaluate the inhibitory potential of the non-pathogenic polypore fungi against G. boninense (G4) via the production of volatile inhibitory organic compounds 49 . The dual plate assay was performed according to Rajani et al. 50 . A mycelia plug (8 mm in diameter) of the potential antagonist was inoculated at the centre of a Petri dish containing PDA (9 cm diameter), incubated for seven days, at 28 °C, and in darkness. Then, the similar-sized mycelial plug of the pathogen, G. boninense (G4), was inoculated onto another PDA plate placed inversely over the PDA plate containing the seven-day-old antagonist, and sealed with parafilm. The VOCs from the antagonist were allowed to fumigate the pathogen. Plates with G. boninense without antagonists served as controls. The setup of the experiment is shown in Fig. 3, and they were arranged in CRD with three replications. The percentage inhibition of diameter growth (PIDG) was calculated using Eq. (3) 51 . where, D1 is the colony diameter of G4 isolate in the control plate, and D2 is the colony diameter of G4 isolate exposed to the antagonist colony. The same inhibition classifications as that for the dual culture assay were adopted.
Statistical analysis. Analysis of variance (ANOVA) was performed, and the means were compared using Tukey's test at a significance level of 0.05. The relationships between the variables were evaluated using Pearson's correlation analysis. Prior to the analysis, the statistical methods were validated in accordance with Zar 52  www.nature.com/scientificreports/ Canada), following the manufacturer's protocol. The fungal internal transcribed spacer (ITS) gene was amplified using the polymerase chain reaction (PCR) with forward universal primer ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) flanking 5′-M13F (5′-GTA AAA CGA CGG CCAGT-3′), and reverse universal primer ITS4 (5′-TCC  TCC GCT TAT TGA TAT GC-3′) flanking 5′-M13R-pUC (5′-CAG GAA ACA GCT ATGAC-3′) 53 . The PCR amplifications were carried out in a 25 μL total reaction volume containing 1 μL gDNA, 0.5 pmol of each primer, deoxynucleotides triphosphates (dNTPs, 200 μM each), a 0.5 U DNA polymerase, PCR buffer, and water. Amplification was performed with an initial denaturation step (98 °C for 2 min), followed by 25 cycles of annealing and extension (98 °C for 15 s; 60 °C for 30 s; 72 °C for 30 s), and a final extension (72 °C for 10 min). The PCR products (1 μL) were run on 1% TAE agarose (agarose with a buffer solution containing a mixture of Tris base, acetic acid and EDTA) at 100 V for 60 min, which yielded 600 to 650 base pairs were purified using standard methods 54 . Sequencing of the purified PCR products was performed using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). The sequence was used to find the homologous sequence via the basic local alignment search tools (BLAST) in the National Center for Biotechnology Information (NCBI) website (https:// www. ncbi. nlm. nih. gov/).

Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files.